Regulation of Gene Expression

Gene Expression Can be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein : (1)  controlling when and how often a given gene is transcribed

(2)  controlling how an RNA transcript is spliced or otherwise processed

(3)  selecting which mRNAs are exported from the nucleus to the cytosol

(4)  selectively degrading certain mRNA molecules

(5)  selecting which mRNAs are translated by ribosomes

(6)  selectively activating or inactivating proteins after they have been made

* most genes the main site of control is step 1: transcription of a DNA sequence into RNA.

Chromatin remodeling

*

* controlling when and how often a given gene is transcribed

!  DNA regulation

!  Chromatin

!  double helix accessibility

!  gene and its surroundings

!  Promoter/Operator (Bacteria)

!  Promoter + enhancing region (Eukaryote )

!  Overview of Eukaryotic gene regulation

Mechanisms similar to those found in bacteria-most genes controlled at the transcriptional level

!  Gene regulation in eukaryotes is more complex than it is in prokaryotes because of:

!  The larger amount of DNA

!  Larger number of chromosomes

!  Spatial separation of transcription and translation

!  mRNA processing

!  RNA stability

!  Cellular differentiation in eukaryotes

Transcription is the Most Regulated Step

!  Transcription; from DNA to RNA, is catalyzed by the enzyme RNA polymerase.

!  Initiation of transcription requires the formation of a complex

between the promoter on the DNA and RNA polymerase. !  Initiation rate is largely controlled by the rate of formation of the

complex DNA (promoter) - RNA polymerase. Rate = number of events per unit time.

Transcriptional Control

!  cis-acting “next to” elements (cis-Regulatory Elements) (CREs) are regions of non-coding DNA which regulate the transcription of nearby genes

!  trans-acting “across from” elements usually considered to be proteins, that bind to the cis-acting sequences to control gene expression. Trans-acting factors affect the expression of genes that may be physically located very far away, even on different chromosomes. The expression of a particular gene may be regulated by the concerted action of both cis and trans-acting elements

The Latin prefix cis translates to “on this side”

“next to”

Promoter: is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites (TSS) of genes, on the same strand and upstream on the DNA (towards the 5 region) Promoters can be about 100–1000 base pairs long

Promoter elements:

!  Core promoter the minimum portion of the promoter required to initiate transcription includes:

•  The transcription start site (TSS) and elements directly upstream

•  A binding site for RNA polymerase

•  General transcription factor binding sites

!  Proximal promoter the proximal sequence upstream of the gene that tends to contain primary regulatory elements, approximately 250 base pairs upstream of the start site

!  Distal promoter (enhancers) the distal sequence upstream of the gene that may contain additional regulatory elements, often with a weaker influence than the proximal promoter

Promoter : regulatory region upstream a gene

1) cis-acting elements:

The transcription start sites or START, is the first nucleotide at which transcription initiates, it means, it is at this site the first nucleotide is incorporated into the transcribed RNA. !  The start site is the first ntds and symbolized by +1, any sequence to the

right of the start is called down stream elements and numbered as +10, +35 and so on.

!  Any nucleotide present on the left of the start is denoted by (-) symbol and the region is called upstream element. The numbers are written as -10, -20, -35 etc.

Bacterial promoters The promoter contains two short sequence elements approximately -10 and -35 nucleotides upstream from the transcription start site •  The sequence at -10 (the -10 element) has the

consensus sequence TATAAT •  The sequence at -35 (the -35 element) has the

consensus sequence TTGACA It should be noted that the above promoter sequences are recognized only by RNA polymerase holoenzyme containing sigma-70

for -10 sequence T A T A A T 77% 76% 60% 61% 56% 82%

for -35 sequence T T G A C A 69% 79% 61% 56% 54% 54%

The holoenzyme is the complete complex containing all the subunits needed for activity.

•  In prokaryotes there are other sequences in the upstream of the promoter, behind –35 sequences. Such sequences may present at

•  –65 to –60 or they may be present at –200 or they may be present at—1000 bp upstream or they may present in downstream regions.

Consensus promoter sequence in E.coli: -35 TCTTGACAT—17+/- 1 — TATAAT-5-8 +1 A(G/T/C) RNA polymerase binding region- -35 --10 +1-----> TTGACA-------TATAAT------- +1-----

Eukaryotic promoters 1) Gene promoters are typically located upstream of the gene and can have regulatory elements several kilobases away from the transcriptional start site (enhancers) •  Located within 100 bp upstream contains TATA box consensus

sequence TATAAA (25 – 30) bases upstream from start point, which is recognized by general transcription factor TATA-binding protein (TBP); and B recognition element (BRE), which is recognized by the general transcription

The AT-rich sequence easy unwinding (because weaker base-stacking interactions among A and T rather than G and C). •  Proximal promoter - the proximal sequence upstream of the gene that

tends to contain primary regulatory elements, approximately 250 base pairs upstream of the start site

!  CAAT box consensus sequence GGCCAATCT that occur upstream by 75-80 bases to the initial transcription site

!  GC box consensus sequence GGGCGG that occur upstream by 110 bases upstream from the transcription initiation site

2) Enhancers & Silencers far away from promoter Distal promoter - the distal sequence upstream of the gene that may contain additional regulatory elements, often with a weaker influence than the proximal promoter

Require the binding of several protein factors to initiate transcription (DNA binding domains on TFs – ‘motifs’)

•  They are called enhancer sequences. Here enhancer means it increases the efficiency of transcription by 100 to 200%. This is achieved through certain proteins bind to enhancer elements, and then contacts RNA holozyme by protein-protein interactions.

Regulation of Transcription

The focal point is whether or not RNA polymerase binds the promoter of a gene and initiates transcription which depends on:

1) Affinity of RNA polymerase for a given promoter

• some promoters are “strong” and bind RNA polymerase with high affinity

• some promoters are “weak” and bind RNA polymerase with low affinity, requiring help from special proteins called transcription factors

• the strength of a promoter depends on its sequence

!  Two consensus sequences of nucleotides in the promoter region.

A. a sequence at -10 (10 bases before the start site): TATAAT

B. a sequence at -35: TTGACA

Regulation Occurs at the Step of Initiation

2 . Structural changes modifying the affinity of the RNA polymerase for the promoter. 3. Influence of proteins referred to as transcription factors, proteins can change the affinity of the RNA polymerase for the promoter. • Proteins that help RNA polymerase bind a promoter (referred to as “activators”) If the proteins increase the affinity, they are activators. • Proteins that inhibit or prevent RNA polymerase from binding a promoter (referred to as “repressors” or “inhibitors”) If the proteins decrease the affinity, they are repressors.

Regulation of Gene Expression

Regulation of Gene Expression

Genes, based on their activity, can be grouped as housekeeping genes and others are classed as induced to express or express in stage specific or tissue specific way.

!  Genes turned on all the time (Constitutive) Housekeeping genes express all the time under all normal conditions.

!  Other genes can be regulated: – Turned On – Turned Off

came originally from the study of how E. coli b a c t e r i a a d a p t t o c h a n g e s i n t h e composition of their growth medium.

Regulation of gene expression allows cells to respond to changes in their

environment

1- An inducible gene is a gene that is expressed in the presence of a substance (an inducer) in the environment. This substance can control the expression of one or more genes (structural genes) involved in the metabolism of that substance. For example, lactose induces the expression of the lac genes that are involved in lactose metabolism. An certain antibiotic may induce the expression of a gene that leads to resistance to that antibiotic.

•  Induction is common in metabolic pathways that result in the catabolism of a substance and the inducer is normally the substrate for the pathway.

2- Repressible gene is those in which the presence of a substance (a co-repressor) in the environment turns off the expression of those genes (structural genes) involved in the metabolism of that substance. e.g., Tryptophan represses the expression of the trp genes.

!  Repression is common in metabolic pathways that result in the biosynthesis of a substance and the co-repressor is normally the end product of the pathway being regulated.

Classification of enzymes by how gene expression is regulated !  Some enzymes are present at all times in a cell

!  These are called constitutive enzymes !  The genes for constitutive enzymes are transcribed at a constant rate because they are

always needed by cells !  RNA polymerase is a good example of a constitutive enzyme

!  Some enzymes (Enzymes of a catabolic pathway) are needed only when a particular compound, such as a nutrient, is available to a cell !  Such enzymes are called inducible enzymes, and the mechanism by which their

synthesis is regulated is called induction !  Genes for inducible enzymes are transcribed only the nutrient becomes available in the

cell's environment

!  Some enzymes (Enzymes of a biosynthetic) are needed only when a particular metabolic intermediate, such as an amino acid, is needed by the cell !  Such enzymes are called repressible enzymes, and the mechanism by which their

synthesis is regulated is called repression !  Transcription of repressible enzymes is shut off if the metabolic intermediate becomes

available to the cell

The Lactose Operon: A Model for Inducible Gene Regulation in Bacteria

•  Best understood cell system for explaining control through genetic induction

Lactose (lac) operon

•  Regulates lactose metabolism in Escherichia coli

•  Three important features: –  The regulator (a gene that codes for a protein capable of repressing

the operon [a repressor]) –  The control locus

•  Promoter- recognized by RNA polymerase •  Operator- a sequence that acts as an on/off switch for transcription

–  The structural locus, made up of three genes each coding for a different enzyme needed to catabolize lactose

β-galactosidase belongs to a group of 3 genes located in close proximity and which are regulated by the same promoter.

Structural genes: Gene Z: β-galactosidase which digest the glycosidic bond. The active form is a tetramer of about 500 kDa. Gene Y: lactose permease which is responsible for the active transport of lactose in the cell. Membrane protein of about 30 kDa. Gene A: b-galactoside transacetylase; transfers the acetyl group from acetyl-CoA to b-galactosides. Dimer of about 60 kDa.

• Operon - series of structural genes all under the control of a Regulatory Gene

Mutations to Characterize the Function of Each of the Genes Found in the Lactose Operon

1.  Mutations in the structural genes:

lacZ mutants: can not metabolize lactose

lacY mutants: can not transport lactose from the extracellular milieu to the intracellular milieu

lacA mutants: no phenotype (no explanations)

2. Mutations in the regulatory genes influence the expression of all 3 structural

genes simultaneously.

•  lac operon is normally turned off •  lac operon is an Inducible operon

Operon consists of: 1. Promoter: region of DNA where RNA polymerase initiates transcription

2. Operator: region of DNA that serves as stop/go signal for transcription

3. Genes: all the ORFs for all the enzymes in the pathway linked end to end; each has its own start and stop codon

4. Terminator: region of DNA where RNA polymerase ends transcription

Metabolism of Lactose in Bacteria

Lactose can�t be used directly by bacteria, it must be degraded into glucose units. The enzyme involved is b-galactosidase

Two Major Types of Transcriptional Control Positive Control: Genes under positive control only be expressed when an

activator protein is present.

Negative control: Genes under negative control are expressed unless a repressor protein is present and blocks their transcription.

!  the levels of different repressors & activators of transcription depend on the cellular environment, which thus determines which genes are ON or OFF!

• Let’s see how this works in genes involved with lactose metabolism in E. coli...

The Lactose Operon Repressor as an Example of Negative Control

The repressor was discovered by Jacob and Monod, who were the first to present a model of gene expression regulation at the molecular level. They obtained the Nobel Prize in 1965 for this model.

Repressor of the Lactose Operon

The lactose operon repressor is coded by the lacI gene, which has its own promoter and terminator sequences.

The repressor molecule is, in fact, a tetramer that binds the operator. The operator sequence overlaps the promoter and the structural genes.

Binding of the Repressor to the Operator

Control of the Binding of the Repressor

The lactose operon is induced when allolactose binds the repressor protein. Allolactose is an inducer. Allolactose is an intermediate in lactose metabolism. Derepression of the lactose operon: binding of allolactose induces a conformational change in the repressor molecule. This change causes a decrease in the affinity of the repressor for the operator sequence on the DNA. The inductor molecule can also cause the repressor to dissociate from the DNA.

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Positive Regulation of Expression of the Lactose Operon

The expression of the lactose operon is not only controlled by the binding of the repressor to the operator site. Even when the repressor is inactivated, transcription of the lactose operon is not optimum. What else is needed to optimize the expression of the lactose operon? Discovery of the activator CAP (catabolic activator protein). CAP binds to the CAP site (located between the i gene and operator site) and increases the affinity of RNA pol for the promoter of the lactose operon. CAP is a dimer and must be activated by cAMP to bind the CAP sequence.

CAP (cAMP binding protein)

CAP activates transcription by increasing the affinity of RNA polymerase for the promoter. CAP as a dimer (two units of 22.5 kDa) that binds the sequence (TGTGA) and an inverted form of this sequence (note that there are variations in the binding site and they may not all contain the inversion). In order for CAP to be able to bind DNA, cyclicAMP (cAMP) must be present. Therefore, cAMP concentrations within the cells will influence transcription of the lactose operon or of other operons under the control of CAP.

The availability of glucose is signaled by intracellular levels of cyclic AMP - When glucose is readily available, cyclic AMP levels in the cell are low - When glucose becomes unavai lable , the ce l l responds by synthesizing additional cyclic AMP

Lactose Operon: Glucose Effect Glucose effect: when lactose and glucose are available, cells preferentially use glucose. Catabolic repression: glucose is a catabolite of lactose and can repress the degradation of lactose. Glucose represses the catabolism of lactose by reducing [cAMPi] and therefore preventing the binding of CAP to DNA.

Basal transcription

Repressor: binds to operator (a region of DNA next to genes)

If something is bound to operator, trasncription cannot occur

Inducer: binds to repressor so it can�t bind to operator Therefore transcription can take place